1 Nichols WJ Jr, Bartelt RJ, Cossé AA, King BH. 2010. Methyl 6-methylsalicylate: a female-produced pheromone component of the parasitoid wasp Spalangia endius. Journal of Chemical Ecology 36:1140-1147. This version matches the text but not the exact formatting of the published article. The final publication is available at www.springerlink.com or by emailing [email protected] Methyl 6-methylsalicylate: A Female-Produced Pheromone Component of the Parasitoid Wasp Spalangia endius (Hymenoptera: Pteromalidae) William J. Nichols Jr.1,* • Allard A. Cossé2 • Robert J. Bartelt2 • Bethia H. King1 1 Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115, USA 2USDA/ARS National Center for Agricultural Utilization Research, Crop Bioprotection Research Unit, 1815 N. University Street, Peoria, Illinois 61604, USA *To whom correspondence should be addressed: e-mail: [email protected] 2 Abstract Sex-pheromone-related behavior and chemistry were studied in the wasp Spalangia endius Walker (Hymenoptera: Pteromalidae), a pupal parasitoid of the house fly, Musca domestica L. (Diptera: Muscidae). Males responded behaviorally to female extracts by arrestment, whereas females did not arrest to male extracts. In a comparison of male and female extracts by gas chromatography-mass spectrometry (GC-MS), two female-specific compounds were found. One was identified as methyl 6-methylsalicylate (gas chromatographic retention time and mass spectrum versus an authentic standard), but the chemical structure of the second compound is still unknown. Male antennae were sensitive to both compounds in electrophysiological tests (GC-EAD). Males responded behaviorally to methyl 6-methylsalicylate by arrestment, but they did not arrest to the second compound. Methyl 6-methylsalicylate has been reported previously from some ant and beetle species, but never from the Pteromalidae. Chemical analysis of the extracts and the male behavioral results are consistent with the hypothesis that methyl 6- methylsalicylate functions as a female-emitted pheromone component at short range, but the exact role of both compounds in intersexual interactions in S. endius remains to be determined. Key Words Gas chromatography-mass spectrometry (GC-MS) • Methyl 6- methylsalicylate • Parasitoid wasp • Pteromalidae • Sex pheromones • Spalangia endius 3 Introduction Pheromones are chemical compounds that are secreted to the outside of insects and elicit responses from conspecifics (Blomquist and Vogt 2003). They are used in many different behavioral contexts, e.g., as attractants or repellants. This study examined pheromone components involved in intersexual interactions in the parasitoid wasp Spalangia endius Walker (Hymenoptera: Pteromalidae). Many aspects of male-female behavioral interactions have been studied in S. endius (King et al. 2005; King and Fischer 2005; King 2006, 2008; Fischer and King 2008; King and Dickenson 2008a, b); yet information on the chemical basis of the behavior has been lacking. Spalangia endius is a widely distributed, 2-3 mm long parasitoid wasp. Its natural hosts are the pupal stage of various flies (Diptera) found in manure and rotting organic matter (Rueda and Axtell 1985). In S. endius, a male's first obvious sexual response to a female begins when he is less than a few centimeters away from her (King et al. 2005). This suggests that at least some pheromone components of S. endius act at short range (King and Dickenson 2008a), although long range components may also exist. Upon detecting a female, the male chases her, often wing fanning (rapidly moving his wings up and down) in the process. Males wing fan to females, but not to males (King 2006), suggesting that males can discriminate between the sexes, probably through the presence of a female-specific pheromone component. Upon contact or near contact, the male either retreats or mounts dorsally. He is especially likely to retreat if she has already mated (King et al. 2005); and retreats appear to be related to females releasing an antiaphrodisiac when a male approaches, rather than from physical aggression from the female (King and Dickenson 2008a). If a male mounts the female, he then continues to court her by vibrating his entire body on her. Fanning, mounting, and vibrating by a male do not require any active solicitation by the female, i.e., even dead females elicit such behaviors. Pheromone components involved in mating behavior in other pteromalid species include pheromones used in attracting a mate and eliciting male courtship behaviors (Ruther et al. 2000). Among insects generally, female-produced pheromones are more common and better known (Keeling et al. 2004); however, in the parasitoid wasp Nasonia vitripennis Walker (Hymenoptera: Pteromalidae), some male pheromones are known to affect a female’s response to the male (e.g., Ruther et al. 2007). It is not uncommon for male and female parasitoids to have many of the same pheromones but in different quantities (Keeling et al. 2004; Ruther and Steiner 2008), and this is true of N. vitripennis (Steiner et al. 2006). Prior to this study no pheromone components of S. endius were known for either males or females. We report the identification of a pheromone component in S. endius that is very different from that reported for other pteromalids. Methods and Materials Spalangia endius Colony The S. endius were from a colony established from wasps collected in 1996 from Zephyr Hills, Florida, USA and maintained by using pupae of a natural host, the house fly, Musca domestica L. (Diptera: Muscidae). Hosts were produced following techniques described in King (1988), but we used wood shavings in 4 place of vermiculite as the rearing substrate. Parasitized hosts were isolated individually in glass test tubes prior to emergence of the wasp in order to obtain female virgin wasps. Virgin males were obtained from separate colonies parasitized by virgin females. Semiochemical Extraction and Gas Chromatography-Mass Spectrometry Analyses Gas chromatography-mass spectrometry (GC-MS) was used to compare semiochemical extracts from males and females to determine if sex-specific compounds were detectable. The semiochemical extracts from males and females were obtained by two different methods, solvent extraction of whole insects and solid phase micro extraction (SPME). A third method (Super-Q trapping of volatiles) was used to prepare extracts from females alone. The purpose of solvent extractions was to remove all soluble compounds from the wasp’s exoskeleton, whereas SPME was used specifically to collect emitted volatiles. For each solvent extraction, ten wasps were killed by freezing at -17oC, transferred to a microtube and extracted in dichloromethane (30 min, 60 µl). Extract volume was reduced to 20 µl under a gentle stream of nitrogen. For SPME, collections were made by exposing the fiber [65 μm polydimethylsiloxane/divinylbenzene blend (Supelco Inc., Bellefonte, PA)] for 20 min to the static headspace of a 1.5 ml vial with a Teflon-lined septum (National Scientific, Rockwood, TX) containing ten insects. Headspace volatiles were collected from females by using Super-Q porous polymer traps (Alltech Associates, Deerfield, IL). This technique permitted a larger quantity of volatiles to be collected than the SPME method. For the trap collection of the volatiles a Magnetek Universal Ser. 41ZF vacuum pump was used to pull the headspace of a 125 ml side-arm flask through 5-mm-diam. glass tubing that contained a 5 to 8-mm plug of Super-Q porous polymer). The Super-Q was held in place by a fine stainless steel screen (fused into trap glass wall) and glass wool. A variable number of 0-d-old females were placed in the flask. Females were replaced with new females every 2 d. Volatiles from a total of 200 females were collected over a 2-mo period. The flask opening was plugged with a cork with a hole, which was filled with glass wool to allow the free inflow of air. To recover the collected volatiles, the Super-Q traps were rinsed weekly with dichloromethane (2 ml). This was concentrated to 400 µl or 2 µl/ female equivalent. No food or water was fed to the insects during the collection period. All three types of extracts were analyzed by a Hewlett Packard 5973 mass selective detector, interfaced with a Hewlett Packard 6890 GC (Agilent Technologies Inc., Santa Clara, CA). A split/splitless inlet was used in splitless mode with either a 15- m DB-1 (0.25 mm i.d., 0.1 µm film thickness ) or a 30-m DB-5MS (0.25 mm i.d., 1.0 µm film thickness) capillary column (J & W, Agilent Technologies Inc., Santa Clara, CA). The oven temperature started at 50oC for 1 min and increased to 280oC at a rate of 10oC/min. Carrier gas was He set at constant pressure (6 psi) and injector temperature was set at 280oC. GC-Electroantennographic Detection (GC-EAD) Analysis Male antennal responses to female-specific compounds from the SPME and Super-Q extracts were examined to determine how sensitively the female-specific compounds were detected. GC-EAD connections were made by inserting the base of an excised male antenna into a saline-filled Ag/AgCl glass pipet grounding electrode. The suspended male antenna was maneuvered by a micromanipulator, into a stream of purified, humidified air (20 ml/sec) 5 that emerged from a 20-cm-long L-shaped glass tube (7 mm i.d.) that ended 1 cm from the antenna. A second glass pipet Ag/AgCl-recording probe (Syntech, Hilversum, The Netherlands), fitted onto a second micromanipulator, was placed in contact with the distal cut end of the antenna. Stimuli were introduced into the air stream from the GC effluent (split between FID and EAD, GC-EAD interface temperature set at 280oC) through a hole in the glass tube positioned 10 cm from the antenna. The GC-EAD responses were amplified (500×) with an AC/ DC UN-6 amplifier (Syntech). Acquisition and analysis of the responses were performed by a computer equipped with an analog to digital conversion board (IDAC, Syntech) running GC-EAD software (Syntech).